Application of active peptide and placenta stem cell exosome for improving physiological characteristics of skin to medicines or cosmetics
A placental stem cell and exosome technology, applied in cosmetics, cosmetic preparations, animal cells, etc., can solve the problems of poor accumulation, poor polypeptide stability, poor cell penetration, etc., to prevent the production, promote proliferation, and promote The effect of fibroblast proliferation
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Embodiment 1
[0028] Embodiment 1 Preparation of placental stem cells
[0029] Isolation of placental mesenchymal stem cells: Cut the 1-2cm thick tissue from the placental chorionic surface, cut into pieces of 1mm×1mm×1mm, rinse with PBS until colorless, and use 0.25% trypsin and 0.2% collagenase IV37 Digest in a water bath at ℃ for 1 hour, oscillate and let stand for 1 minute to make the liquid naturally divide into three layers, absorb the suspension of the middle layer into a centrifuge tube, add PBS, shake well and centrifuge at 700 r / min for 5 minutes, discard the supernatant, and add 3 mL of mesenchyme Stem cell culture medium (87% DMEM, 10% fetal bovine serum (FBS), 1% L-glutamine, 1% penicillin and streptomycin) was resuspended, centrifuged at 700r / min for 5min, and the supernatant was discarded. Add appropriate amount of culture medium and mix well, transfer to cell culture flask, 37°C, 5% CO 2 , when the cultured cells grew to 80% in the incubator, trypsinization was performed fo...
Embodiment 2
[0031] Example 2 Preparation of highly active placental mesenchymal cell stem cell exosomes
[0032] Take the placental stem cells prepared in Example 1, and when the cell confluence grows to 85%, replace the exosome secretory medium (15% AGS, 0.5% ginseng saponins plus 84.5% DMEM / F12) for 60 hours, and collect the cell supernatant . Centrifuge at 300×g for 10min to remove dead cells and large cell debris, centrifuge at 2000×g for 10min to remove dead cells and cell debris, centrifuge at 10000×g / min for 30min to remove larger vesicles of cell debris, filter through a 0.22 μm syringe filter, Remove microvesicles and possible apoptotic bodies. Use a 20mL empty needle to transfer the supernatant to an ultracentrifuge tube, 10 6 Centrifuge at × g for 70 min, remove the supernatant, collect the precipitate, and obtain the crudely extracted exosomes, 10 6 Centrifuge at × g for 70 min, remove the supernatant, and dissolve the precipitate with 100 μL PBS to obtain relatively pure e...
Embodiment 3
[0034] Example 3 Preparation of placental mesenchymal stem cell exosomes control example
[0035] The placental stem cells prepared in Example 1 were taken, and when the cell confluence grew to 85%, the exosome secretion-stimulating medium (15% AGS plus 85% DMEM / F12) was changed to culture for 60 h, and the cell supernatant was collected. Centrifuge at 300×g for 10min to remove dead cells and large cell debris, centrifuge at 2000×g for 10min to remove dead cells and cell debris, centrifuge at 10000×g / min for 30min to remove larger vesicles of cell debris, filter through a 0.22 μm syringe filter, Remove microvesicles and possible apoptotic bodies. Use a 20mL empty needle to transfer the supernatant to an ultracentrifuge tube, 10 6 Centrifuge at × g for 70 min, remove the supernatant, collect the precipitate, and obtain the crudely extracted exosomes, 10 6 Centrifuge at × g for 70 min, remove the supernatant, and dissolve the precipitate with 100 μL PBS to obtain relatively pu...
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